A Unique Mechanism of Glycine-Specific Inhibition of Bacterial Translation by Bottromycin A₂

---

Inna A. Volynkina^1,2,a*, Aleksandr A. Grachev³, Alexei Livenskyi^2,4,5, Daria K. Yagoda⁵, Pavel S. Kasatsky³, Olga A. Tolicheva³, Ekaterina S. Komarova^1,6, Alexey E. Tupikin⁷, Vera A. Alferova^6,8, Anastasiia O. Karakchieva¹, Arina A. Nikandrova^2,6,9, Mikhail V. Biryukov⁹, Yuliya V. Zakalyukina¹⁰, Lubov V. Dorofeeva¹¹, Yuriy A. Ikhalaynen¹, Igor A. Rodin¹, Dmitrii A. Lukianov^1,2, Marsel R. Kabilov⁷, Alena Paleskava^3,12, Andrey L. Konevega^3,12,13,b*, Petr V. Sergiev^1,2,6,c*, and Olga A. Dontsova^1,2,6,8

¹⁰Department of Soil Science, Lomonosov Moscow State University, 119234 Moscow, Russia

¹¹All-Russian Collection of Microorganisms (VKM), G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Scientific Center for Biological Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Oblast, Russia

¹²Institute of Biomedical Systems and Biotechnologies, St. Petersburg Peter the Great Polytechnic University, 195251 Saint Petersburg, Russia

¹³Centre for Nano, Bio, Info, Cognitive, and Social Sciences and Technologies (NBICS Center), National Research Center “Kurchatov Institute”, 123182 Moscow, Russia

¹Department of Chemistry, Lomonosov Moscow State University, 119234 Moscow, Russia

²Center for Molecular and Cellular Biology, Skolkovo Institute of Science and Technology, 121205 Moscow, Russia

³Molecular and Radiation Biophysics Division, Petersburg Nuclear Physics Institute named by B. P. Konstantinov of National Research Center “Kurchatov Institute”, 188300 Gatchina, Leningrad Region, Russia

⁴Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

⁵Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia

⁶A. N. Belozersky Research Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

⁷Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Novosibirsk Oblast, Russia

⁸Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

⁹Department of Biology, Lomonosov Moscow State University, 119234 Moscow, Russia

^* To whom correspondence should be addressed.

Received: October 18, 2025; Revised: November 6, 2025; Accepted: November 6, 2025

---

The rise of antimicrobial resistance among pathogenic bacteria poses a critical challenge to modern medicine, highlighting an urgent need for novel therapeutic agents. Bottromycin A₂ (BotA2) is a promising candidate for future drug development, demonstrating potent activity against clinically relevant pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus, and Mycoplasma species, although its molecular mechanism of action has remained unclear until now. Here, we demonstrate that BotA2 inhibits bacterial translation with unique context specificity determined by the mRNA coding sequence. Using high-throughput toe-printing coupled with deep sequencing (Toe-seq analysis), we show that BotA2 induces ribosome pausing predominantly when a glycine codon enters the A-site of the ribosome, regardless of the codon identities in the P- and E-sites. Our biochemical and biophysical data indicate that BotA2 specifically arrests glycine-delivering ternary complexes on the ribosome, thereby preventing full accommodation of incoming Gly-tRNA^Gly within the peptidyl transferase center. Altogether, our findings uncover a previously undescribed mechanism of translation inhibition, driven by the context-specific immobilization of ternary complexes on elongating ribosomes.

---

KEY WORDS: bottromycin, antibiotics, translation, inhibition of protein synthesis, context specificity, ternary complex

DOI: 10.1134/S0006297925603740

Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

---